Pluripotent stem cells have attracted attention because of having the property of being capable of differentiating into various cells constituting the body and the property of being capable of maintaining their characteristics being undifferentiated, and are not only applied to drug discovery screening and elucidation of disease mechanisms but also under world-wide study as a material for regenerative medicine.
The world's first phase 1 clinical trial using human ES cells started against acute spinal-cord injury in the U.S.A in 2010; furthermore, an investigational new drug (IND) application for phase ½clinical trials using human ES cells against retinal degenerative disease was approved by FDA; and regenerative medicine research using human pluripotent stem cells continues rapid development.
Particularly, iPS cells as new human pluripotent stem cells originating in Japan have great advantage that they have a low ethical roadblock because of, for example, no use of fertilized embryos and can be established also from autologous tissue, and thus they are receiving high expectations from the field of regenerative medicine. In Japan, Riken Center for Developmental Biology, Institute of Biomedical Research and Innovation Laboratory, and other institutes plan to start clinical studies using iPS cells with age-related macular degeneration patients in fiscal 2013, and Keio University also intends to start clinical studies in spinal cord injury patients in 2015.
As the clinical application of human pluripotent stem cells such as ES cells and iPS cells are started as just described above, a system to supply cells by securing quality and safety is not sufficiently developed. For pluripotent stem cells, the preparation method, culture conditions, storage conditions, and the like affect qualities such as characteristics being undifferentiated, differentiation potency, and proliferative capacity. Thus, managements not based on an appropriate method may produce results different for each producer and each user. This becomes a cause of bringing negative effects such as the decreased reliability of stem cell therapy and the occurrence of health hazards due to the therapy. Thus, there are necessary a maintenance culture method high in reliability and reproducibility and a measurement/evaluation system.
For example, although pluripotent stem cells are not directly used but used after differentiating them into desired cells for transplantation in a cell therapy, it has been pointed out that if a cell source having differentiated into desired cells is contaminated with undifferentiated cells, these undifferentiated cells become a cause of tumorgenesis. Accordingly, there is a need for the development of a technique for evaluating whether cells to be used for cell therapy are contaminated with undifferentiated cells, i.e., tumorgenic cells.
In contrast, somatic stem cells, which are various compared to human pluripotent stem cells including ES cells and iPS cells, have been in clinical application as established techniques. However, it is not easy to stably obtain cells having quality suitable for transplantation; thus, it represents a very important challenge to establish a quality verification method for mesenchymal stem cells and a stable culture method based thereon. There is also a need for the development of a quality verification method for cells before transplantation in evaluating the effectiveness of cell transplantation using somatic stem cells, understanding the mechanism thereof, and evaluating risk.
Previously, the present inventors exhaustively analyzed the sugar chain profiles of human iPS cells (114 specimen) prepared from 5 types of different somatic cells (skin, fetal lung, endometrial membrane, placental artery, and amniotic membrane) and human ES cells (9 specimen), using lectin microarray.
As a result, despite the different sugar chain profiles of the original somatic cells for each tissue, it was found that all of the prepared iPS cells showed almost the same sugar chain profile and the introduction of reprograming genes caused uniform convergence into sugar chain structures analogous to those of ES cells. According to the results of analyzing the lectin array data of human ES/iPS cells and human somatic cells in detail, the expression level of α2-6Sia, α1-2Fuc, and type 1 LacNAc was presumed to be markedly increased in undifferentiated human ES/iPS cells compared to in somatic cells. In addition, rBC2LCN was found to bind only to undifferentiated human ES/iPS cells by expression analysis of glycosyltransferase genes using DNA array and a method using a mass spectrometer (Non Patent Literature 1).
The rBC2LCN described above is a recombinant BC2LCN lectin (YP_002232818) that corresponds to the N-terminal domain of the BC2L-C protein derived from a gram-negative bacterium (Burkholderia cenocepacia), and is expressed in transformed Escherichia coli, and is a lectin recognizing the sugar chain structures “Fucα1-2Galβ1-3GlcNAc” and “Fucα1-2Galβ1-3GalNAc” in the nonreducing terminus of a complex sugar chain (Non Patent Literatures 1 and 3).
In the above-described experiment using the lectin array, the present inventors found that rBC2LCN reacted with undifferentiated human ES/iPS cells but completely failed to react with differentiated somatic cells (skin, fetal lung, endometrial membrane, placental artery, and amniotic membrane). It is construed that rBC2LCN specifically reacts with the sugar chain structures “Fucα1-2Galβ1-3GlcNAc (=H type 1 structure)” and “Fucα1-2Galβ1-3GalNAc (=H type 3 structure)” having 2 (α1-2Fuc and typel LacNAc) of “α1-2Fuc”, “typel LacNAc”, and “α2-6Sia”. These two sugar chain structures are sugar chains highly expressed on human ES/iPS cells and hardly expressed on differentiated cells of the skin, fetal lung, endometrial membrane, placental artery, and amniotic membrane.
This indicates that the sugar chain ligand recognized by rBC2LCN is a novel undifferentiation sugar chain marker characterizing undifferentiated cells and also indicates that rBC2LCN can be used as a probe specific for the undifferentiation sugar chain markers “Fucα1-2Galβ1-3 GlcNAc” and/or “Fucα1-2Galβ1-3GalNAc” (hereinafter, both are sometimes together referred to as “Fucα1-2Galβ1-3GlcNAc/GalNAc”).
Thereafter, the team of Drukker et al. also found that an antibody recognizing “Fucα1-2Galβ1-3GlcNAc” recognizes ES and iPS cells in an undifferentiated state (Non Patent Literature 2), supporting the above findings of the present inventors.
However, the antibody of Drukker et al. specifically reacts with “Fucα1-2Galβ1-3GlcNAc (=H type 1 structure)” but does not react with “Fucα1-2Galβ1-3GalNAc (=H type 3 structure)”. When the antibody is compared with rBC2LCN, it cannot detect “Fucα1-2Galβ1-3GalNAc” or “sugar chains containing Fucα1-2Galβ1-3GalNAc” in undifferentiated cells; thus, the antibody has a disadvantage of having sensitivity not sufficiently increased when compared with rBC2LCN of the present inventors.